Prolactin (PRL) has numerous and diverse actions associated with lactation and reproduction as well as with somatotropic and osmoregulatory activities. PRL release is subjected to tonic inhibition by hypothalamic and posterior pituitary dopamine. Indirect evidence also suggests the existence of a PRL-releasing factor (PRF). Although several peptides, e.g. TRH, VIP and oxytocin, have been postulated as physiological PRFs, none satisfies all the criteria to subserve this role. Recent in vivo and in vitro studies from our laboratory demonstrated the presence of a potent PRF in the posterior pituitary whereas the hypothalamus has only little PRF activity. PRF appears to be a small peptide(s) which participates in the suckling-induced and proestrus- associated, rise in PRL. The initial goal of this proposal is to isolate PRF from posterior pituitary extracts and identify its structure. Subsequently, synthetic PRF and antibodies raised against it will be used to determine the functional role of PRF in the overall regulation of PRL secretion. The first specific aim will employ an in vitro bioassay and will focus on the characterization, isolation and purification of PRF by means of specific proteolytic enzymes and fractionation on both preparative and analytical HPLC. The second specific aim will focus on the structural determination of PRF by amino acid sequencing and mass spectrometry. Following its synthesis by a solid phase method, polyclonal antibodies against PRF will be generated and an RIA will be established. The third specific aim will validate synthetic PRF as a physological modulator of PRL release. Its biological activity will be verified both in vivo and in vitro, and the importance of endogenous PRF will be assessed by passive immunization with PRF antibodies. PRF release will be studied by means of electrical stimulation of a hypothalamo-posterior pituitary explant in a compartmentalized chamber. The fourth specific aim will utilize RIA for PRF and will determine whether PRF originates in the hypothalamus or within the pituitary proper. The distribution of PRF in hypothalamic and extrahypothalamic sites will be explored and its release into the systemic circulation will be investigated. These studies will extend our understanding of the control of PRL secretion and should contribute to the diagnosis and management of PRL-secreting tumors.